1. Field of the Invention
The present invention relates, in general, to nuclear receptors and, in particular, to the Constitutive Androstane Receptor (CAR; NR 1I3) and to a method of identifying ligands therefor.
2. Background Information
Nuclear receptors are members of a superfamily of ligand-modulated transcription factors that mediate responses to a variety of agents including steroids, retinoids and thyroid hormones (see Beato et al, Cell 83:851 (1995); Kastner et al, Cell 83:859 (1995); Mangelsdorf et al, Cell 83:841 (1995)). Nuclear receptors have characteristic sequence motifs, for example, a variable amino terminal domain containing an autonomous activation function critical for cell and target specificity, a more carboxy terminal central region that contains a DNA binding domain and a distal carboxy terminal ligand binding domain (LBD). These motifs make it possible to isolate new nuclear receptor family members on the basis of sequence homology alone (Giguere et al, Nature 331:91 (1998)). Since isolation does not require prior identification of the receptor ligand, this technology has led to the discovery of multiple xe2x80x9corphanxe2x80x9d receptors. Like receptors with known ligands, these orphan receptors contain sequence motifs characteristic of ligand binding domains. Thus, ligands may ultimately be found for these orphan receptors. Since nuclear receptors have importance as drug targets, the discovery of selective ligands for orphan receptors represents an important step towards finding novel pharmacological intervention pathways.
One member of the steroid/retinoid/thyroid hormone nuclear receptor superfamily of ligand-activated transcription factors, the CAR (originally named MB67), has been implicated in mediating the effects of xenobiotics on cytochromes P450 (CYPs) 2B and 3A (CYP2B and CYP3A, respectively) gene expression (Kliewer et al, Science 284(5415):757-760 (1999), Savas et al, Mol. Pharmacol. 56:851-857 (1999), Waxman, Archives of Biochemistry and Biophysics 369(1):11-23 (1999)). CAR, a member of the nuclear receptor subfamily NR1, is most abundantly expressed in liver and has strong constitutive activity in cell-based reporter assays in the absence of any added ligand (Baes et al, Mol. Cell. Biol. 14:1544-1552 (1994), Choi et al, Journal of Biological Chemistry 272(38):23565-26571 (1997)). In HepG2 cells or other cell lines, exogenously expressed CAR can enter the nucleus and regulate the expression of reporter constructs. This constitutive activity can be inhibited by superphysiological concentrations of the testosterone metabolites androstanol and androstenol (Forman et al, Nature 395:612-615 (1998)). These androstanes inhibit the interaction of CAR with the coactivator protein SRC-1, suggesting that xe2x80x98deactivationxe2x80x99 is mediated by direct binding to the orphan receptor. In contrast to transfected cell lines, CAR is not present in the nucleus of primary hepatocytes but is instead sequestered in the cytoplasm. Treatment of primary hepatocytes with either phenobarbital (PB) or the planar hydrocarbon 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) results in the translocation of CAR into the nucleus, where it binds to its cognate DNA response elements as a heterodimer with the 9-cis retinoic acid receptor (RXR) and activates the transcription of target genes, including retinoic response elements (Baes et al, Mol. Cell. Biol. 14:1544-1552 (1994)) and CYP2B (Honkakoski et al, Mol. Cell. Biol. 18:5652-5658 (1998), Kawamoto et al, Molecular and Cellular Biology 19(9):6318-6322 (1999), Sueyoshi et al, Journal of Biological Chemistry 274(10):6043-6046 (1999)). CAR/RXR binding sites have been identified in the PB-responsive regions of the mouse, rat, and human CYP2B genes. The effects of PB on CYP2B expression are blocked by the phosphatase inhibitor okadaic acid (Kawamoto et al, Molecular and Cellular Biology 19(9):6318-6322 (1999)), suggesting that dephosphorylation of CAR, rather than direct ligand binding, is involved in its translocation into the nucleus.
The present invention provides a method of identifying CAR ligands. Such ligands can be used to elucidate the function of CAR, a potential drug discovery target, including its role in multicomponent regulatory networks. Additionally, this assay can be used to determine selectivity of ligands of other nuclear receptors, i.e., as a negative assay. Such an assay can be used to distinguish specific ligands for other nuclear receptors from non-specific ligands. Furthermore, this assay can be utilized as a secondary assay to a cell based CAR assay to verify that an effect seen in the cell based assay is directly on CAR. Additionally, this assay can be used to confirm that the effect of a compound in a fluorescence based cofactor assay is due to that compound binding at the binding pocket of CAR by virtue of its competitive displacement of clotrimazole.
The present invention relates to the nuclear receptor CAR and to a method of identifying ligands therefor. The method comprises assaying putative CAR ligands for their ability to displace clotrimazole from CAR LBD-containing proteins.
The present invention provides a method of screening a selected compound for its ability to inhibit the binding of clotrimazole to a Constitutive Androstane Receptor (CAR) ligand binding domain (LBD)-containing polypeptide comprising:
i) contacting said selected compound and clotrimazole with said CAR LBD-containing polypeptide under conditions such that clotrimazole can bind to said CAR LBD-containing polypeptide in the absence of said selected compound, and
ii) determining the amount of clotrimazole bound to said CAR LBD-containing polypeptide and comparing that amount to an amount of clotrimazole bound to said CAR LBD-containing polypeptide in the absence of said selected compound,
wherein a reduction in the amount of clotrimazole bound to said CAR LBD-containing polypeptide in the presence of said selected compound indicates that said selected compound inhibits the binding of clotrimazole to said CAR LBD-containing polypeptide.
The present invention further relates to a compound identifiable using the above method as being capable of inhibiting the binding of clotrimazole to CAR LBD. The instant invention further relates to a composition comprising such an identifiable compound.
The present invention additionally provides a method of determining whether a ligand for a selected nuclear receptor is non-specific for the selected nuclear receptor, comprising (a) contacting a ligand that binds the selected nuclear receptor with clotrimazole and a Constitutive Androstane Receptor (CAR) ligand binding domain (LBD)-containing polypeptide under conditions such that clotrimazole can bind to the CAR LBD-containing polypeptide in the absence of the ligand, and (b) determining the amount of clotrimazole bound to the CAR LBD-containing polypeptide, wherein an amount of clotrimazole bound to the CAR LBD-containing polypeptide that is less than that bound to the CAR LBD-containing polypeptide in the absence of ligand indicates that the ligand inhibits binding of clotrimazole to the CAR LBD-containing polypeptide and is non-specific in its binding to the selected nuclear receptor.
Further, the instant invention provides a method of screening a selected compound for ability to bind a human Constitutive Androstane Receptor (hCAR) ligand binding domain (LBD) comprising (a) contacting the selected compound and clotrimazole with the hCAR LBD-containing polypeptide under conditions such that clotrimazole can bind the hCAR LBD-containing polypeptide in the absence of the selected compound, and (b) determining the amount of clotrimazole bound to the hCAR LBD-containing polypeptide, wherein an amount of clotrimazole bound to the hCAR LBD-containing polypeptide that is less than that bound to the hCAR LBD-containing polypeptide in the absence of the selected compound indicates that the selected compound binds the hCAR LBD.
Objects and advantages of the invention will be clear from the description that follows.